PROJECT SUMMARY This proposal is to continue our studies of glycoprotein (GP)Ib-IX-V complex that is essential to platelet physiology. Initially identified as a platelet sensor for flow shear stress through its interaction with von Willebrand factor (VWF), GPIb-IX-V plays a critical role in hemostasis and thrombosis. It is also tapped for additional roles in inflammation, platelet genesis and clearance. Malfunction of this multi-subunit receptor complex can lead to severe bleeding diathesis and contribute to many cardiovascular diseases. During the current funding period we have identified, for the first time, a mechanosensory domain (MSD) in the juxtamembrane region of the GPIb? subunit. The MSD unfolds upon tension-mediated pulling on an engaged A1 domain of VWF or anti-GPIb? antibody. Furthermore, unfolding of the MSD induces signaling through GPIb-IX, resulting in accelerated platelet clearance and thrombocytopenia. Our study on MSD has initiated a new paradigm for the mechanosensing and activation mechanism of GPIb-IX-V. First, neither the affinity nor the binding epitope of an anti-GPIb? antibody is the key to GPIb-IX activation. Instead, a tensile force generated by the bound antibody is what is needed to unfold the MSD and activate GPIb-IX. Second, antibody-mediated platelet crosslinking may be a common feature for soluble ligands to generate tension on GPIb-IX and to activate it. Third, activation of GPIb-IX by plasma VWF or anti-GPIb? antibodies results in receptor desialylation and exposure of ?-galactose residues on the platelet surface, which have been shown in recent studies to mediate platelet clearance. These findings, along with our preliminary observations under adherent conditions, suggest the existence of hitherto unidentified elements modulating the mechanical dynamics and function of the MSD. To continue our investigation of the structure-function of GPIb-IX-V, we propose here to identify and characterize the elements activating and modulating the MSD with multidisciplinary approaches in three Specific Aims. Aim 1 is to define the structural and mechanical elements of physiological ligand VWF that are required to activate GPIb-IX. Aim 2 is to identify and characterize residues and novel modulatory elements in the MSD that are critical to its dynamics and function. Aim 3 is to explore the mechanical regulation of GPIb-IX activity by GPV through their interaction. Completion of the proposed study will critically advance the fundamental mechanosensing and mechanoregulatory mechanism of the GPIb-IX-V complex and the platelet. It will also establish a solid structural foundation for understanding the diverse functional roles of GPIb-IX-V in platelet clearance, hemostasis, and thrombosis.